1. Field of the Invention
The present invention relates generally to plant growth and development and more specifically to identification of novel plant kinases and methods of identifying compounds that modulate the activity of such kinases in plants.
2. Description of the Related Art
Phosphorylation by protein kinases is one of the most common and important regulatory mechanisms in signal transmission. Plant genomes encode many protein kinases. Some of the plant kinases are homologues of kinases found in animals and fungi, while others have novel structures. Kinases comprise the largest known protein group, a super family of enzymes with widely varied functions and specificities. Kinases covalently modify proteins and peptides by the attachment of a phosphate group to one or more sites on the substrate protein.
Many of the known protein kinases use adenosine triphosphate (ATP) as the phosphate donor and place the gamma phosphate from the ATP onto an acceptor amino acid. The amino acids that can act as acceptors of the gamma phosphate group are Serine (Ser), Theronine (Thr), Tyrosine (Tyr), or Histidine (His). The majority of protein kinases known can be categorized as either Ser/Thr kinases or Tyr kinases. The Histidine kinases, originally identified in bacterial two-component systems, have recently been found as part of signaling pathways in plants and fungi.
Protein kinases can be further subdivided into families based on the amino acid sequences surrounding or inserted into the kinase catalytic domain. The function of these sequences surrounding the catalytic domain have been shown to allow regulation of the kinase as it recognizes its target substrate protein (Hardie, G and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.).
The primary structure of the kinase catalytic domain is conserved and can be further subdivided into 11 subdomains. Each of these 11 subdomains contains sequence motifs that are highly conserved or invariant (Hardie, G and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.). One such motif found in kinases identified to date is an invariable domain comprising asparagine, phenylalanine and glycine residues (DFG domain) surrounded by several conserved residues (Hunter T., (1997) Philos Trans R Soc Lond B Biol Sci. 353(1368):583-605, Hunter T. (1995) Cell. 80(2):225-36; van der Geer P, et al. (1994) Annu Rev Cell Biol. 10:251-337).
Studies have shown that kinases are key regulators of many cellular functions, such as: cell proliferation, cell differentiation, signal transduction, transcriptional regulation, cell motility, and cell division. Few, if any physiological processes exist in eukaryotes that are not dependent on phosphorylation.
Of particular importance in intracellular signaling are the mitogen-activated protein kinases (MAPKs). MAPKs are also members of the Ser/Thr family of protein kinases. MAPKs play a central role in the transduction of diverse extracellular stimuli, including signals that regulate development and differentiation, into intracellular responses in yeast and animals cells via phosphorylation cascades. Homologues of the MAPKs found in animals and yeast have been found in plants. Previous studies have suggested the involvement of MAP kinase cascades in the regulation of auxin signaling. In vitro phosphorylation of a bacterially produced Arabidopsis MAP kinase by a tobacco cell extract is three to four-fold more effective after treatment of protoplasts with the synthetic auxin 2,4-D, as compared to extracts from auxin-starved cultures (Mizoguchi et al. (1994) Plant J. 5:111-122). The importance of MAP kinase phosphorylation has also been demonstrated by over-expression in maize protoplasts of the catalytic domain of the tobacco MAPKK kinase NPK1, which blocks transcription from the auxin-responsive GH3 promoter (Kovtun et al. (1998) Nature 395:716-720). A role for protein phosphorylation in auxin transport has also been inferred from the discovery that the Arabidopsis gene ROOTS CURL IN NPA 1 (RCN1) encodes a regulatory subunit of protein phosphatase 2A (Garbers et al. (1996) EMBO J. 15:2115-2124).
Plant growth and development are governed by complex interactions between environmental signals and internal factors. Light regulates many developmental processes throughout the plant life cycle, from seed germination to floral induction (Chory, J. Trends Genet., 9:167, 1993; McNellis and Deng, Plant Cell, 7:1749, 1995), and causes profound morphological changes in young seedlings. In the presence of light, hypocotyl growth is inhibited, cotyledons expand, leaves develop, chloroplasts differentiate, chlorophylls are produced, and many light-inducible genes are coordinately expressed. It has been suggested that plant hormones, which are known to affect the division, elongation, and differentiation of cells, are directly involved in the response of plants to light signals (P. J. Davies, Plant Hormones: Physiology, Biochemistry and Molecular Biology, pp 1-836, 1995; Greef and Freddericq, Photomorphogenesis, pp 401-427, 1983). The interactions between phototransduction pathways and plant hormones however are not well understood.
Auxin is one of the classical plant hormones and regulates many aspects of plant development, including cell division, cell elongation and cell differentiation in both the root and the shoot of plants (M. Estelle, Bioessays 14:439-44, 1992 and L. Hobbie et al., Plant Mol. Biol. 26:1499-519, 1994). For example, apical dominance as well as lateral root growth are under auxin control, and manipulation of auxin signaling can be used to affect growth of both the shoot and the root system.
From its point of synthesis at the plant apex (Davies, P. J. (1995) The plant hormones: Their nature, occurrence, and functions. In Plant Hormones: Physiology, Biochemistry and Molecular Biology, P. J. Davies, ed. (Netherlands: Kluwer Academic Publishers), pp. 1-12), the phytohormone auxin is directionally transported through the plant body to effect an astonishing variety of morphological processes. Auxin is required early in development to establish the bilateral axis of the developing embryo Hadfi et al. (1998) Development 125:879-887). Later, auxin participates in vascular element patterning and differentiation (Aloni, R. (1995). Biochemistry and Molecular Biology, P. J. Davies, ed. (Netherlands: Kluwer Academic Publishers), pp. 531-545), lateral organ outgrowth in the root and shoot (Okada et al. (1 991) Plant Cell 3:677-684; Celenza et al. (1995) Genes Dev. 9:2131-2142), and local growth responses to external stimuli such as light and gravity (Kaufman et al. (1995). Hormones and the orientation of growth. In Plant Hormones: Physiology, Biochemistry and Molecular Biology, P. J. Davies, ed. (Netherlands: Kluwer Academic), pp. 547-570).
While an understanding of the mechanisms of auxin action at the molecular level is preliminary, genetic and biochemical approaches have begun to reveal discrete aspects of auxin transport, signaling and response. Two related Arabidopsis proteins, PINFORMED (PIN) and ETHYLENE INSENSITIVE ROOT 1 (EIR1)/AGRAVITROPIC1 (AGR1)/PIN2, which share homology with bacterial membrane transporters, function as auxin efflux carriers in the shoot and root, respectively (Chen et al. (1998) Proc. Natl. Acad. Sci. USA 95:15112-15117; Gxc3xa4lweiler et al. (1998) Science 282:2226-2230; Luschnig et al. (1998) Genes Dev. 12:2175-2187; Mxc3xcller et al. (1998) EMBO J. 17:6903-6911). The auxin influx carrier AUXIN INSENSITIVE 1 (AUX1), which shares homology with plant and fungal amino acid permeases, functions in root gravitropism (Bennett et al. (1996) Science 273:948-950; Marchant et al. (1999) EMBO J. 18:2066-2073). TRANSPORT INHIBITOR RESPONSE 3 (TIR3) has been implicated in auxin transport in both the root and the shoot. tir3 mutants have fewer binding sites than wild type for the auxin transport inhibitor NPA (naphthylphthalamic acid), suggesting that the TIR3 gene product either encodes or regulates an NPA binding protein (Ruegger et al. (1997) Plant Cell 9:745-757). An auxin receptor has not been unambiguously identified, but overexpression of the auxin-binding protein ABP1 affects cell expansion, a transcription-independent auxin-regulated process (Jones et al. (1998) Science 282:1114-1117).
Apart from proteins involved in auxin transport and binding, several classes of auxin-signaling molecules have been identified. One class includes regulators of protein stability such as AUXIN RESISTANT 1 (AXR1) and TIR1. These two proteins are components of the RUB conjugation pathway, which is analogous to and converges with the ubiquitin pathway (Lammer et al. (1998) Genes Dev. 12:914-926; Liakopoulos et al. (1998) EMBO J. 17:2208-2214). TIR1 is an F-box protein that together with SKP1 and Cdc53 constitutes an E3 ubiquitin ligase complex (Patton et al. (1998) Genes Dev. 12:692-705; Gray et al. (1999) Genes Dev. 13:1678-1691). The only identified target for RUB modification is the Cdc53 subunit of the E3 complex itself, the functional significance of which is unknown (Lammer et al., 1998; Liakopoulos et al., 1998).
Another class of auxin signaling molecules comprises the ARF (Auxin Response Factor) and Aux/IAA families of transcription factors (Guilfoyle et al. (1998) Plant Physiol. 118:341-347). ARFs bind to auxin response elements through an N-terminal DNA binding domain, while the carboxy-terminus contains two protein-protein interaction domains that are also found in the Aux/IAA family of early auxin-response genes. In vitro homodimerization and heterodimerization within each family, as well as interactions between the families, suggest that combinatorial action of these proteins confers cell or tissue specificity in auxin response (Kim et al. (1997) Proc. Natl. Acad. Sci. USA 94:11786-11791; Ulmasov et al. (1997a) Science 276:1865-1868; Ulmasov et al. (1997b) Plant Cell 9:1963-1971; Ulmasov et al. (1999) Plant J. 19:309-319). Although ARFs and Aux/IAAs were initially identified by biochemical methods, several Arabidopsis mutants with defects in auxin signaling have subsequently been shown to carry lesions in ARF or Aux/IAA genes (Hardtke and Berleth (1998) EMBO J. 17:1405-1411; Rouse et al. (1998) Science 279:1371-1373; Tian and Reed (1999) Development 126:711-721). One example is the MONOPTEROS (MP) gene, which encodes ARF5 and whose loss of function in the shoot results in a pin-like inflorescence similar to that seen in pin mutants, or in plants treated with polar auxin transport inhibitors (Okada et al., 1991; Berleth and Jurgens (1993) Development 118:575-587).
The invention disclosed herein is based on the discovery that an otherwise conserved motif in protein kinases is modified in various plant kinases. This conserved motif, the xe2x80x9cDFDxe2x80x9d domain, has been found in several plant kinases and is termed the xe2x80x9cDFGxe2x80x9d domain. One such exemplary plant kinase is identified in the present invention the PINOID (PID) gene, which encodes an auxin signaling and/or response protein.
PID gene encodes a member of a newly identified plant-specific serine-threonine protein kinase family. PID encodes a protein kinase, but belongs to a different class of serine-threonine kinases than the MAP, MAPK, or MAPKK kinases. PID contains 13 of the 14 invariant residues found in fungal and metazoan protein kinases (Hanks et al., 1988). The single exception is the replacement by aspartate of the invariant glycine of the DFG motif found in the consensus sequence of catalytic subdomain VII. The DFD motif of PID is identified in the present application as being present in several other putative plant kinases. At least one of these kinases, NPH1, functions in blue-light mediated phototropism and encodes a functional kinase (Christie et al. (1998) Science 282:1698-1701).
In contrast to PIN and MP, which are expressed in the vasculature of the inflorescence stem as well as in developing primordia. This expression pattern is PID is predominantly expressed in anlagen of lateral primordia, consistent with PID affecting downstream events in auxin signaling. In contrast to pin and mp mutants, which have substantially decreased auxin flow in the inflorescence, pid mutants show only a modest reduction in polar auxin transport (Okada et al., (1991); Bennett et al. (1995) Plant J. 8:505-520; Przemeck et al. (1996) Planta 200:229-237). Moreover, PID overexpression results in shoot and root phenotypes similar to those of auxin-insensitive mutants, indicating that PID regulates auxin signaling.
One embodiment of the invention disclosed herein provides an assay for identifying plant kinases, specifically, DFD kinases. The plant kinase assay includes screening nucleic acid obtained from a plant in order to identify a nucleotide sequence that encodes a contiguous DFD amino acid sequence in a region of the nucleotide sequence ordinarily known to encode a xe2x80x9cDFGxe2x80x9d sequence. The presence of a DFD catalytic domain, in addition to the presence of other kinase structural markers, indicates that the nucleic acid encodes a plant kinase, or a fragment thereof.
In another embodiment, the invention provides methods for identifying compounds that modulate the activity of DFD plant kinases. The kinase assays of the invention include incubating at least one putative kinase-modulating compound and a DFD plant kinase polypeptide containing a DFD motif under conditions sufficient for the components to interact, and determining kinase activity in the presence and the absence of the compound, wherein a difference in activity is indicative of a compound that modulates kinase activity of a plant DFD kinase. An exemplary DFD kinase that can be utilized in the method of the invention is a PINOID polypeptide. Compounds or agents, including small molecules, peptides or nucleic acid sequences, that affect DFD kinase activity by binding to a DFD kinase can be identified by the method of the invention. A complex between a DFD kinase and a test compound or agent can be identified and the binding agent isolated therefrom. Such compounds or agents can be further evaluated to determine whether they bind and/or affect DFG kinase activity.
In another embodiment, there are provided methods for modulating the level or activity of DFD kinases, comprising exposing DFD kinases to DFD inhibitors. In a preferred embodiment of the present invention, there are provided methods for inhibiting the growth or exterminating plants expressing DFD kinases. Preferably, the inhibitor will bind DFD kinases, more preferably, the inhibitor will be specific for DFD kinases and will not bind or affect DFG kinases. Such inhibitors are especially useful in methods for selectively growing plants expressing kinases containing the DFG domain.
In another embodiment, the invention provides a method for producing a genetically modified plant characterized as having early or increased loss of apical dominance, such as increased branching and/or lateral root growth, as compared to a wild-type plant. The method includes transferring at least one copy of a DFD kinase-encoding polynucleotide operably associated with a promoter to a plant cell to obtain a transformed plant cell and producing a plant from the transformed plant cell. Such genetically modified plants may exhibit increased branching and/or lateral root growth. An exemplary DFD kinase includes members of the PINOID gene family.
In another embodiment, the invention provides a method for genetically modifying a plant cell such that the plant produced from the cell is characterized phenotypically by increased branching and/or lateral root growth as compared to a wild-type plant. The method comprises contacting a plant cell with at least one copy of a DFD kinase-encoding polynucleotide to obtain a transformed plant cell and growing the transformed plant cell under plant forming conditions to obtain a plant having increased shoot architecture or root growth.
In another embodiment, the invention provides a method for obtaining a genetically modified plant characterized as having increased apical dominance (e.g., decreased branching and/or lateral root growth) as compared to a wild-type plant. The method comprises transferring at least one loss-of-function dominant negative variant of a DFD kinase-encoding polynucleotide, operably associated with a promoter, to a plant cell to obtain a transformed plant cell and producing a plant from the transformed plant cell having increased apical dominance (i.e., due to overexpression of the loss-of-function variant in the plant).
In yet another embodiment, the invention provides a method for producing a plant characterized as having increased branching and/or lateral root development yield by contacting a plant having an endogenous DFD kinase gene operably linked to its native promoter, with a promoter-inducing amount of an agent which induces DFD kinase gene expression, wherein induction of DFD kinase gene expression results in production of a plant having increased branching and/or lateral root development as compared to a plant not contacted with the inducing agent. For example, transcription factors or chemical agents may be used to increase expression of DFD kinase in a plant, in order to provide plants having increased branching for more homogeneous fruit maturation, dwarf varieties, grass with little need of mowing, and the like.